The endocrine system is a network of glands that produce and release hormones that help control many important body functions, including the body's ability to change calories into energy that powers cells and organs. The endocrine system influences how the heart beats, how the bones and tissues grow, even one's ability to reproduce. It plays a vital role in whether or not a person develops diabetes, thyroid disease, growth disorders, sexual dysfunction, and a host of other hormone-related disorders.
When the endocrine glands malfunction by under producing or releasing the essential hormones or when the glands are destroyed or removed, e.g. due to cancer, it would then be necessary to replace the missing essential hormones.
The derivation of pluripotent stem cells into endocrine tissues (PSC-derived endocrine tissues), such as pancreatic islets or thyroid follicles, provides particularly attractive opportunities to replace the missing essential hormones in vivo in the afflicted persons. PSC-derived endocrine tissues can achieve the envisioned in vivo function without orthotopic transplantation because the engraftment of these hormone-secreting tissues in any location with access to circulating blood would potentially achieve function and even clinical rescue from a variety of common endocrine diseases, including diabetes mellitus and hypothyroidism. Indeed, the differentiation of PSCs into pancreatic islet-like cells has produced cells capable of secreting insulin in vivo following transplantation (Cheng et al., 2012; Pagliuca et al., 2014).
Recent progress in the differentiation of PSCs in vitro has allowed the derivation of desired cell lineages and in some instances, the in vitro self-assembly of the differentiated lineage cells into 3D structures, referred to as organoids (Lancaster and Knoblich, 2014; McCracken et al., 2014). However, transplantation and in vivo function of these engineered cells have been less successful, typically due to poor engraftment or failure to structurally integrate orthotopically transplanted PSC-derived cells into native recipient tissues.
Thyroid epithelial cells have been engineered from stem cells in two separate approaches: (1) by forced, over-expression of integrated genes encoding multiple transcription factors, NKX2.1 and PAX8, in the stem cells (Antonica et al., 2012), and (2) by growing the stem cells in growth factor supplemented media (Longmire et al., 2012). In marked contrast to the poor in vivo efficacy of the stem cell-derived organoids above described, the thyroid epithelial cells generated to date from PSCs through the forced over-expression of integrated genes encoding multiple transcription factors displayed in vivo functional potential (Antonica et al., 2012).
Without resorting to the over-expression of integrated genes encoding multiple transcription factors, the in vitro directed differentiation of PSCs into thyroid epithelial cells using growth factor supplemented media, however, do not produce the same outlook as forced over-expression PSC-derived engineered cells. This method only resulted in immature PSC-derived engineered cells that fail to express the full genetic program necessary for either iodine metabolism or functional thyroid hormone biosynthesis (Arufe et al., 2006; Arufe et al., 2009; Jiang et al., 2010; Longmire et al., 2012; Ma et al., 2009). In Longmire et al. for example, the six-factor cocktail serum-free media was sufficient to direct initial differentiation of embryonic stem cell (ESC)-derived definitive endodermal cells to enter the thyroid lineage. This directed differentiation was assessed by the expression of thyroid epithelial-specific genes, such as thyroglobulin (Tg). However, these thyroid lineage cells did not progress further into the thyroid differentiation pathway; these cells did not display full thyroid maturation as the genes encoding all proteins required for iodine metabolism and thyroid hormone biosynthesis were not robustly expressed, such as the sodium iodine symporter (Nis) and thyroid peroxidase (Tpo).
The primary hurdle preventing the successful differentiation of PSCs into mature thyroid cells has been a lack of knowledge of the signaling pathways that regulate early thyroid embryonic development. “Directed differentiation” of PSCs utilizes sequential exposure of undifferentiated PSCs to a series of growth factor-supplemented media designed to recapitulate the sequence of developmental milestones that normally occurs during the embryonic differentiation of a desired cell lineage. This approach, based on the observation that PSCs resemble the inner cell mass of the developing blastocyst embryo, has been employed to successfully produce a wide variety of non-thyroid lineages from PSCs (Murry and Keller, 2008), but the derivation of functional thyroid follicular epithelial cells has not yet been achieved. The derivation of thyroid epithelial cells via directed differentiation remains a compelling goal given the known capacity of thyroid epithelia, once mature, to self-organize and form follicular structures in vitro. Indeed trypsinized thyroid follicular epithelial cells from developing or adult animals have been shown to self-aggregate and form follicles during in vitro culture (Hilfer et al., 1968; Mallette and Anthony, 1966; Martin et al., 1993). Moreover, recent work has demonstrated that thyroid cells generated from ESCs through the forced over-expression of transcription factors also formed follicles in vitro or after transplantation in vivo (Antonica et al., 2012; Ma et al., 2015).